1. Field of the Invention
The present invention relates to oligonucleotide chip composition and a manufacturing method thereof, and in particular, to an oligonucleotide chip composition for analyzing hepatitis C virus (HCV) genotype and detecting method thereof.
2. Description of the Prior Art
In general, hepatitis C virus (hereinafter, referred to as ‘HCV’), which is a kind of hepatitis virus, is a principal factor in causing serious diseases such as hepatitis including acute hepatitis and chronic hepatitis, which may develop into hepatic cirrhosis and hepatoma. The HCV is infected via blood transfusion and fluid (Choo et al., Science 244, 359–362, 1989). It is estimated that about 4 hundred million people over the world are infected with the HCV: 0.2–2% of people in the developed countries such as Europe, North America and Japan; 2–5% in South America and Asia; over 5% in Africa; 1.6% in Korea (Park et al., J. Viral Hepat. 2, 195–202, 1995). The HCV is a very threatening virus to human health, and it is not convalescent unlike hepatitis B virus (HBV). 50–85% of the infected people develope on chronic hepatitis. Because the HCV is RNA virus, scientists have not developed any proper remedies and vaccines, much less basic study on the HCV yet. Because diagnosis methods are developed by the development of molecular biology, people can avoid the possibility of infection resulting from blood transfusion. However, because route of infection is still obscure, the possibility of infection still remains.
The HCV is positive-single strand linear RNA virus consisting of about 9,500 bases and about 3,000 amino acids, having a size of 50 nm which Choo et al. discovered in non-A or non-B (NANB) type hepatitis virus obtained from blood plasma of chimpanzee in 1989. The HCV basically consists of open reading frame (ORF) producing structural protein of core, nucleocapsid and envelope glycoprotein and unstructural protein of helicase, viral protease, RNA-dependent RNA polymerase, transcriptase and regulatory peptide (Chou et al., Jpn. J. Med. Sci. Biol. 4, 147–157, 1991). Both ends of ORF has 5′-untranslated region (5′UTR) and 3′UTR, respectively. 5′UTR, which is a best preserved portion in HCV gene, has about 340 bp and a stem-loop structure (Han et al., Proc. Natl. Acac. Sci. U.S.A. 88, 1711–1715, 1991).
Because of high mutation rate of HCV, (1.44–1.92)×10−3 of base replacement in HCV is generated per year. 5′UTR and capsid of HCV gene are best preserved. Mutation generates most frequently in E1 and E2 (Ogata et al., Proc. Natl. Acad. Sci. U.S.A. 88, 3392–3396, 1991). Because of this property, HCV shows high gene polymorphism. Thus, up to now HCV is classified into 6 types and subtypes ranging from several to tens (Simmonds et al., J. Gen. Virol. 74, 2391–2399, 1993; Cha et al., Proc. Natl. Acad. Sci. U.S.A. 89, 7144–7148, 1992). Because there has been no standard method of classifying these types, researchers use different classification but Simmond's classification is generally used. This classification attaches figures (1, 2, 3, . . . ) on genotype and alphabet letters (a, b, c, . . . ) on subtypes. According to the classification, HCV shows 31–35% difference between genotypes, 20–23% difference between subtypes, and 1–10% difference within even the same subtypes (Simmonds et al., J. Gen. Virol. 74, 661–668, 1993).
A method for analyzing HCV genotype is used in identifying infection of HCV and prognosticating infection course and treatment effect of IFN-α (Hino et al., J. Med, Virol. 42, 299–305, 1994). And the method for analyzing genotype is used in examining distribution and vaccine development because it shows different distribution according to the area and race (Greene et al., J. Gen. Virol. 76, 211–215, 1995). IFN-α is the most common antiviral agent for treating HCV. IFN-α is effective over 50% of the patients. However, only 25% of the patients may return to normally functioning liver and have no HCV in blood serum. Here, according to the study about relation between IFN-α treatment effect and HCV type, the IFN-α shows excellent treatment effect in genotypes 1a, 2, 3 and 5 but low treatment effect in genotypes 1b and 4 (Yoshioka et al., Hepatology 16, 293–299, 1992). In addition, genotypes 1b and 4 is rapidly transformed into chronic hepatitis while genotypes 1a and 2a is improved for the better symptom (Lopez-Labrador, et al., J. Hepatol. 27, 959–965, 1997).
General methods for analyzing HCV genotype are as follows. First, SSP-PCR method is to use PCR primers having specific for HCV genotype. The method is to perform RT-PCR by combining PCR primers specific for various genotypes in the core region. The method has an advantage that results can be obtained right after RT-PCR, but the method has disadvantages that new type can not be analyzed if mutations are generated around a recognition site of primers. Thus, the SSP-PCR method is not desirable in analyzing HCV genotype having diverse mutations. Second, PCR-RFLP method is to amplify a 5′UTR region by RT-PCR and use restriction enzyme (Park et el., J. Med. Microbiol. 47, 1998). PCR-RFLP method has an advantage that results can be easily and simply obtained, but HCV genotype are not analyzed unless the used restriction enzyme recognizes mutation region. Third, a method is to amplify a 5′UTR region by RT-PCR and hybridize an oligonucleotide probe specific on a nitrocellulose (NC) membrane for HCV genotype. According to the method, the precise results can be obtained by kinds of probe, but there is a limit in fixing a number of probes on NC membrane. In addition, the method spends a lot of time and labor in handling and analyzing various specimens because only a genotype of a person can be analyzed in the NC membrane.